Men with prostate cancer suffer significant impairments in quality of life not only from the disease itself but also as a consequence of treatment. Novel approaches for prevention of prostate cancer are still desirable because: (a) many risk factors associated with this disease are not easily modifiable; and (b) a clinically viable preventive intervention to reduce disease-related cost, morbidity, and mortality associated with prostate cancer is lacking. The overall goal of this research project is to develop cruciferous vegetable constituent phenethyl isothiocyanate (PEITC) for prevention of prostate cancer. In the previous funding period, we demonstrated, for the first time, that PEITC is bioavailable in the prostate of mice following dietary administration. More importantly, dietary PEITC administration resulted in a significant decrease in incidence and burden of poorly-differentiated prostate cancer in a transgenic mouse model (TRAMP). Other noteworthy published findings from the previous funding period germane to this application include: (a) PEITC treatment induces apoptotic as well as autophagic cell death in prostate cancer cells, and both of these processes are intimately linked to production of reactive oxygen species (ROS); (b) the underlying molecular circuitry of PEITC-induced apoptosis downstream of ROS generation involves Bax activation, whereas impairment of oxidative phosphorylation (OXPHOS) due to inhibition of complex III of the mitochondrial electron transport chain is the trigger for ROS production; and (c) these effects of PEITC are not observed in a normal human prostate epithelial cell line (PrEC). We are equally excited with our findings that PEITC treatment inhibits glycolysis (reversal of Warburg effect) in prostate cancer cells, but not in PrE cells. However, the in vivo significance of these cellular observations demonstrating bioenergetics inhibition by PEITC is still unclear. Likewise, the molecular mechanism(s) by which PEITC inhibits glycolysis or OXPHOS remains elusive. This revised renewal application outlines well-designed and hypothesis-driven experiments to not only address the above identified mechanistic gaps in our knowledge but also to determine the efficacy of PEITC for prevention of prostate cancer in a preclinical model (Hi-Myc transgenic mouse model) closely mimicking the kinetics and the biology of human prostate cancer. The Hi-Myc mouse model is well-suited for the studies proposed in this application because: (a) PEITC treatment decreases protein and mRNA levels of c- Myc leading to inhibition of its transcriptional activity in a panel f human prostate cancer cells; (b) activity of probasin promoter, which drives the c-Myc transgene expression in the prostate of Hi-Myc mice, is not affected by PEITC treatment; (c) PEITC inhibits viability of a cell line established from tumor of a Hi-Myc mouse; and (d) both the Warburg effect and OXPHOS are regulated by c-Myc. Our published results and unpublished preliminary observations led us to hypothesize that PEITC inhibits prostate cancer development in Hi-Myc mouse model in association with suppression of glycolysis and OXPHOS. The specific aims of the revised renewal application include: (1) Determination of the effect of dietary PEITC administration on incidence and burden of prostatic intraepithelial neoplasia (PIN) and adenocarcinoma in the dorsal, lateral, and ventral prostate lobes of Hi-Myc mice; (2) Elucidation of the mechanism underlying PEITC-mediated suppression of c-Myc expression and glycolysis; and (3) Determination of the mechanism by which PEITC inhibits OXPHOS. Translational Impact: Specific aim 1 not only establishes effectiveness of PEITC for prevention of prostate cancer in a clinically-relevant mouse model but also provides animals and tissues for in vivo validation of the cellular mechanistic observations demonstrating glycolysis and OXPHOS inhibition by PEITC. Specific aims 2 and 3 are developed to gain insights into the molecular circuitry of bioenergetics inhibition by PEITC. Together, these studies may lead to identification of biomarker(s) (e.g., non-invasive in vivo measurement of tumor glycolysis by 1H-MRSI) potentially useful in future clinical trials. In vivo validated pharmacodynamic biomarker(s) are essential for clinical development of PEITC because cancer incidence is too demanding of an end point for malignancies with long latency such as prostate cancer. Biomarker(s) predictive of PEITC tissue exposure and possibly response are not yet known. Completion of the proposed preclinical studies provides knowledge critical for design of a future pilot biomarker-driven trialin prostatectomy patients in a neoadjuvant window setting.